Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates

Abstract

BackgroundAdeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression.MethodsThree female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression.ResultsMacaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system.ConclusionsThese results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.

Liver Gene Therapy Approaches for Acute Intermittent Porphyria: Metabolic Correction and Immunological Hurdles

“…92,99,123 Most probably, the PBGD transgene protein will not elicit immune responses in a clinical trial for AIP.…”

“…Additionally, human capsid-specific T cells were found to produce interferon-γ, a cytokine that upregulates the major histocompatibility complex I (MHC I) molecules that are critical for CD8 + target cell recognition, whereas those from NHP T cells were less efficient in this regard. 108 Coadministration of an immunosuppression protocol that included mycophenolate mofetil (MMF) with rAAV vector does not induce toxicity in NHP and does not meaningfully improve rAAV5-mediated gene expression 123 (Figure 4). However, Montenegro-Miranda et al 124 demonstrated that in Gunn rats MMF inhibited the synthesis of the complementary DNA strand following entry of the rAAV into the target cells.…”

“…A different study performed in mice has shown a remarkably deleterious effect of MMF on transgene expression when the drug is initiated one month after vector administration. 123 This intriguing MMF-dependent drug-mediated interference with transgene expression takes place via an as yet elusive mechanism which requires a functional adaptive immune system.…”

“…As a consequence, repeated gene transfer with the same rAAV5 vector was impossible. 123 Interestingly, under abrogation of antibody responses, the persistence of circulating rAAV5 antigens is dramatically long-lasting under the strong immunosuppressive regimen and neutralizing antibodies are elicited following drug cessation. This can be interpreted as B cells and their production of antibodies are important for rAAV antigen clearance.…”

“…Antithymocytic globulin (ATG), MMF, methylprednisolone, tacrolimus (FK506), and rituximab were given in combination during 12 weeks before the rAAV5 administration to block T-and B-cell-mediated adaptive immune responses in NHP. 123 The rationale behind the choice of the strong immunosuppressive regimen was its effectiveness in a previous study in curtailing the immune response toward first-generation adenoviral vectors in macaques. 127 However, the humoral response against the rAAV5 capsid was only partially reduced under rituximab treatment, and it reached the values obtained in the control animals once the immunosuppression protocol was halted.…”